Protein achieving improved blocking efficiency

ABSTRACT

It is intended to provide a method of conveniently screening a novel protein or a novel partial sequence protein having a blocking ability based on amino acid sequence data; and a protein achieving an improved blocking efficiency that can be expressed on a large scale in  Escherichia coli . A method of screening a novel protein or a novel partial sequence protein having a blocking ability based on amino acid sequence data; a protein characterized by achieving an improved blocking efficiency owing to an amino acid sequence modification; and a method of utilizing the protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is the U.S. national phase of InternationalPatent Application PCT/JP04/09785, filed on Jul. 2, 2004, which claimsthe benefit of Japanese Patent Application No. 2003-191081, filed onJul. 3, 2003.

TECHNICAL FIELD

The present invention relates to a method of screening a novel proteinor a novel partial sequence protein candidate for blocking having ablocking ability based on amino acid sequence data, and further relatesto a protein achieving an improved blocking efficiency by modifying anamino acid sequence of a protein. The present invention also relates toa blocking reagent, a stabilizing agent, an excipient, a protein foldingaccelerator, a protein refolding accelerator and a coating agent formedical use, which contain the protein. The present invention canimprove the blocking efficiency of a protein capable of being expressedon a large scale in Escherichia coli, and is useful when a recombinantprotein excellent in blocking efficiency is produced on a large scale.

BACKGROUND ART

Conventionally, proteins directly extracted from living body componentshave been often used as blocking agents used for immunoassays. Inparticular historically, albumin and casein derived from bovine havebeen widely used. However, recently, various limitations have occurredowing to problems such as mad cow disease. Meanwhile, method ofproducing them using recombinant technology has an advantage that apathogen (substance) can be excluded, but is virtually off frompractical use due to problems such as productivity.

Accordingly, it can be said that an attempt to use a protein derivedfrom Escherichia coli as an alternative protein is preferable in termsof productivity. However, there has been a problem in that a proteincapable of being expressed on a large scale does not always have a goodblocking efficiency and a protein which can be produced by recombinanttechnology and is excellent in blocking efficiency must be found.

Recently, gene sequences in various organisms have been elucidated, buta principle to find a protein excellent in blocking efficiency by apredicted amino acid sequence has not be known yet, and it has seemedthat it takes a lot of trouble to search such a protein.

Due to such reasons, an alternative protein which can be produced on alarge scale by recombinant technology and is excellent in blockingefficiency has been required.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing a concept of measuring a blocking efficiency.

FIG. 2 is a view showing a bisection method of a protein.

FIG. 3 is a view showing a blocking mechanism of a protein having ablocking ability.

FIG. 4 is a view showing a three dimensional structure of DnaK 384-607.A β-sheet portion and an α-helix portion correspond to an N terminus anda C terminus, respectively.

FIG. 5 is a view showing blocking efficiencies of various DnaK mutants.In FIG. 5,

Blank: no blocking

384-638: DnaK 384-638

384-607: DnaK 384-607

384-578: DnaK 384-578, a mutant where a part of the α-helix structurewas deleted.

384-561: DnaK 384-561, a mutant where about a half of the α-helixstructure was deleted.

508-607: DnaK 508-607, a mutant where the β-sheet portion was deleted(composed of the α-helix).

525-607: DnaK 525-607, a mutant where the β-sheet portion and a part ofthe α-helix were deleted (composed of the α-helix).

BSA: BSA fraction V

FIG. 6 is a view showing structures of an Escherichia coli DnaK proteinand produced mutants thereof.

FIG. 7 is a view showing a three dimensional structure of DnaK 381-553.

FIG. 8 is a view showing a blocking mechanism of a protein in which ahydrophobic domain was modified.

FIG. 9 is a view showing blocking efficiencies of various DnaK mutants.

384-607: DnaK 384-607

384-607 (VAV): DnaK 384-607 (D479V, S481V)

419-607: DnaK 419-607

BSA: BSA fraction V

Blank: no blocking

FIG. 10 is a view showing a correlation of DnaK mutant concentrationsand blocking effects.

FIG. 11 is a view showing blocking speeds of DnaK mutants.

FIG. 12 is a view showing practical application of the DnaK mutant toblocking in ELISA.

FIG. 13 shows content rates of hydrophilic and hydrophobic amino acidsin proteins frequently used for blocking and a highly hydrophobicprotein.

FIG. 14 shows characteristics of amino acids contained in the N terminalside and C terminal side of BSA, α-casein, lipase and DnaK 384-607. |Δ|represents an absolute value of a difference betweenhydrophilic/hydrophobic rates in the N terminal side and the C terminalside.

FIG. 15 shows comparison of blocking effects of a (native) DnaK fragmentwithout histidine tag. BSA (fraction V) and the DnaK 419-607 fragmentwere prepared at concentrations of 10 mg/mL and 0.5 mg/mL, respectively,and shown as the comparison at high concentrations. BSA (fraction V) andthe DnaK 419-607 fragment also were prepared at concentrations of 2mg/mL and 0.1 mg/mL, respectively, and shown as the comparison at lowconcentrations.

FIG. 16 is a view showing data at high concentrations in FIG. 15.

FIG. 17 is a view showing data at low concentrations in FIG. 15.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method of easilyfinding a protein having a blocking ability from an amino acid sequence,as well as provide a protein which can be produced in Escherichia colion a large scale and achieves an improved blocking efficiency bymodification of the amino acid sequence.

Based on the above context, as a result of an extensive study, acharacter concerning an amino acid sequence characteristic for a proteinhaving a blocking ability has been found, as well as it has been foundthat a blocking efficiency can be dramatically enhanced by modifying anamino acid sequence of the protein found in such a way to complete thepresent invention.

That is, the present invention is composed of the followingconstitution.

[1] A method of screening a novel protein or a novel partial sequenceprotein candidate for blocking having a blocking ability based on aminoacid sequence data, the method of screening the protein or the partialsequence protein, which meets the following conditions:

-   A) the amino acid sequence of the protein is divided into two, and    an absolute value of a difference between hydrophilic/hydrophobic    rates in divided two portions is 0.1 or more, calculated using the    following formula from content rates of hydrophilic amino acids (D,    E, K, H, R, Y) and hydrophobic amino acids (G, A, V, L, I, M, F, W,    P); [Hydrophilic/hydrophobic rate]=[Content rate of hydrophilic    amino acids]/[Content rate of hydrophobic amino acids];-   B) the hydrophilic/hydrophobic rate in a hydrophilic portion (a    higher value of hydrophilic/hydrophobic rate) is 0.5 or more; and-   C) the protein is composed of more than 100 amino acid residues.

[2] A novel protein or a novel partial sequence protein for blockinghaving a blocking ability, screened by the method of [1], the protein orthe partial sequence protein which can be obtained by the followinganalysis step D and meets the following condition E;

-   D) (1) a step of adding a candidate protein (0.5 to 1 mg/mL, diluted    with 20 mM Tris-HCl, pH 7.0) which meets the conditions of [1] and    bovine serum albumin (fraction V) prepared by the same way to    respective wells of a polystyrene immunotiter plate, blocking at 2    to 10° C. for 4 to 5 hours and removing solutions;-   (2) a step of adding normal human serum diluted 25 to 100 times with    PBS(−), leaving stand at 37° C. for one hours, and subsequently    washing the plate with PBS(−) (0.05% TWEEN® (polysorbate) 20); and-   (3) a step of comparing IgG amounts non-specifically absorbed to the    plate using an enzyme-labeled anti-human IgG antibody by a    calorimetric method using a chromogenic substrate; and-   E) the absorbance for the candidate protein is 2.5 times or less    than the developed color intensity for bovine serum albumin.

[3] A novel protein or a novel partial sequence protein for blockinghaving a blocking ability, screened by the method of [1], the protein orthe partial sequence protein which can be obtained by the followinganalysis step F and meets the following condition G;

-   F) (1) a step of dissolving horseradish peroxidase for labeling at    0.05 mg/mL in a candidate protein solution (0.5 to 1 mg/mL, diluted    with PBS(−)) and a bovine serum albumin (fraction V) solution    prepared by the same way;-   (2) a step of dispensing the above diluted solutions to a    polystyrene 96-well microplate;-   (3) a step of leaving stand at 25° C. for one hour, removing the    solutions and washing with PBS(−) containing 0.02% TWEEN®    (polysorbate) 20;-   (4) a step of adding a tetramethylbenzidine solution, incubating at    37° C. and subsequently adding 1N sulfuric acid to stop a reaction    and develop a color; and-   (5) a step of measuring the absorbance by a microplate reader; and-   G) the absorbance for the candidate protein is 2.5 times or less    than the developed color intensity for bovine serum albumin.

[4] A novel protein or a novel partial sequence protein for blockinghaving a blocking ability, screened by the method of [1], the protein orthe partial sequence protein which can be obtained by the followinganalysis step H and meets the following condition I;

-   H) (1) a step of adding a candidate protein (0.5 to 1 mg/mL, diluted    with 20 mM Tris-HCl, pH 7.0) which meets the conditions of [1] and    bovine serum albumin (fraction V) prepared by the same way to    respective wells of a polystyrene immunotiter plate, blocking at 2    to 10° C. for 4 to 5 hours and removing solutions;-   (2) a step of adding a peroxidase solution prepared at 0.05 mg/mL,    leaving stand at 37° C. for one hour and subsequently washing the    plate with PBS(−) (0.05% TWEEN® (polysorbate)20);-   (3) a step of leaving stand at 25° C. for one hour, subsequently    removing the solution and washing with PBS(−) containing 0.02%    TWEEN® (polysorbate)20; and-   (4) a step of adding a tetramethylbenzidine solution, incubating at    37° C. and subsequently adding 1N sulfuric acid to stop a reaction    and develop a color; and-   (5) a step of measuring the absorbance by a microplate reader; and-   I) the absorbance for the candidate protein is 2.5 times or less    than the developed color intensity for bovine serum albumin.

[5] A novel protein achieving an improved blocking efficiency bymodifying an amino acid sequence of a protein or a partial sequenceprotein which meets the conditions A, B and C according to [1].

[6] The protein achieving the improved blocking efficiency according to[5] characterized in that the amino acid sequence is modified by aminoacid substitution, deletion and insertion.

[7] The protein achieving the improved blocking efficiency according to[5] characterized by being derived from a prokaryotic organism or aneukaryotic organism.

[8] The protein achieving the improved blocking efficiency characterizedby being derived from an “HSP 70 family protein”.

[9] The protein achieving the improved blocking efficiency according to[8] characterized by being derived from a DnaK protein.

[10] The protein achieving the improved blocking efficiency according to[8] characterized by being a protein obtained by deleting a part of anamino acid sequence of the DnaK protein.

[11] The protein achieving the improved blocking efficiency according to[8], which is a protein obtained by deleting a part of an amino acidsequence of the DnaK protein, characterized in that an amino acidsequence from an N terminus to at least position 387 and at mostposition 472 has been deleted.

[12] The protein achieving the improved blocking efficiency according to[8], which is a protein obtained by deleting a part of an amino acidsequence of the DnaK protein, characterized in that an amino acidsequence from an N terminus to at least position 387 and at mostposition 418 has been deleted.

[13] The protein according to [8] composed of an amino acid sequence ofpositions 419 to 607 in the DnaK protein.

[14] The protein achieving the improved blocking efficiency according to[8] characterized in that a part of hydrophilic amino acids issubstituted with hydrophobic amino acids in the DnaK protein wherein anATPase domain or a part thereof has been deleted.

[15] The protein achieving the improved blocking efficiency according to[8] which is a protein wherein a part of an amino acid sequence isdeleted in the DnaK protein wherein an ATPase domain or a part thereofhas been deleted, wherein aspartic acid at positions 479 and 481 in theamino acid sequence is substituted with valine.

[16] The protein achieving the improved blocking efficiency according to[8] composed of an amino acid sequence of positions 384 to 607 in theDnaK protein, wherein aspartic acid at positions 479 and 481 in theamino acid sequence has been substituted with valine.

[17] A protein for blocking having one or more hydrophilic domains andone or more hydrophobic domains, wherein the hydrophobic domain can beabsorbed to a material surface and the hydrophilic domain can cover thehydrophobic domain absorbed to the material surface.

[18] A modified protein characterized in that a blocking speed isfurther enhanced than that of BSA.

[19] The modified protein according to [18] characterized in that ablocking ability in less than 10 minutes is more excellent than that ofBSA under a condition where protein amounts are adjusted so as toexhibit a blocking efficiency equivalent to that of BSA in blocking for3 hours.

[20] The protein according any of [2] to [19] characterized by having atag sequence.

[21] The protein according to [20] characterized in that the tagsequence is selected from a histidine tag, a maltose binding protein(MBP) tag, a glutathione S-transferase (GST) tag, a Flag tag, a Myc tag,and a tandem affinity purification tag.

[22] The protein according to any of claims [2] to [19] characterized inthat an optional amino acid sequence is added.

[23] A method of producing a protein characterized by producing theprotein according to any of [2] to [22] using a prokaryotic organism.

[24] A method of producing a protein characterized by producing theprotein according to any of [2] to [22] using Escherichia coli.

[25] A method of producing a protein characterized by producing theprotein according to any of [2] to [22] using a cell-free proteinsynthesis method.

[26] A method of purifying the protein according to any of [2] to [22],characterized by passing through a heating step.

[27] A method of using the protein according to any of [2] to [21] forblocking, stabilization, size enlargement, protein folding promotion,protein refolding promotion, coating and medical use.

[28] A blocking reagent, a stabilizing agent, an excipient, a proteinfolding accelerator, a protein refolding accelerator, a coating agentfor cell attachment or a coating agent for medical use, which containsthe protein according to any of [2] to [22].

BEST MODES FOR CARRYING OUT THE INVENTION

As used herein, blocking refers to preventing a component fromnon-specifically absorbing to a vessel or a carrier, and in particularrefers to preventing a protein from non-specifically absorbing to aresin such as plastic. In various measurements, it is problematic thatthe component subjected to the measurement is non-specifically absorbedto a material surface, which prevents the measurement as a background.Particularly in an immunoassay, an antibody is remarkably absorbed to apolystyrene plate. Typically, a manipulation where the protein easilyabsorbed to the resin is previously added to prevent non-specificabsorption of the antibody, i.e., the blocking is widely performed. Inthe immunoassay, it is essential to physically absorb the protein to thematerial surface, and thus, the plate made from the resin such aspolystyrene, to which the protein is easily absorbed is often used, butan extra component is also remarkably absorbed. Therefore actually, agood blocking agent is always required. In clinical diagnostic drugs, itis often problematic that an enzyme for the diagnostic drug isnon-specifically absorbed to a cell in an automatic analyzer.

In the present invention, with immunoassays in mind, the blocking effectwas measured by measuring the non-specific absorption of human serum IgGknown as an example of the most remarkable non-specific absorption tothe polystyrene plate. That is, the measurement is composed of thefollowing steps.

-   (1) A protein solution whose blocking ability to be tested is added    to a polystyrene 96-well plate, and left stand for a certain time    period to block the plate.-   (2) The solution in (1) is removed, diluted human serum is added and    incubated.-   (3) The plate is washed, subsequently a labeled anti-human IgG    antibody is added and incubated.-   (4) After the plate is washed, amounts of non-specifically bound IgG    are compared by a calorimetric method.    The detailed method will be described below. This method was very    effective when the present invention was performed. An outline of    the method is shown in FIG. 1.

As a blocking agent, conventionally bovine serum albumin (BSA) andbovine milk casein have been widely used. However, it has not beenexplained theoretically what nature of the protein their high blockingability is derived from, and generally it is a common belief thathydrophobicity of the protein causes the high binding ability.

Thus first, for the purpose of examining whether a highly hydrophobicprotein has the blocking ability, lipase derived from Pseudomonasaeruginosa, known for having high contents of hydrophobic amino acidswas used as the blocking agent to examine its blocking ability. Lipaseis used for clinical laboratory tests, but it has been known that itsabsorbing ability to a plastic surface is high and the absorption to thecell upon measurement is problematic. However, as a result of theexperiment, it was demonstrated that the blocking effect was scarcelyobserved. Rates of hydrophilic amino acids and hydrophobic amino acidsof respective proteins were shown in FIG. 13. Comparing them, it isfound that the hydrophobicity of BSA and casein is lower than that oflipase. It is discussed from this that a phenomenon where the protein isabsorbed owing to its hydrophobicity is related to the blocking abilitybut this relation is not explain all cases. As a reason why the blockingability of lipase was not exerted, it is speculated that the protein wasnon-specifically absorbed to lipase absorbed to the material surface.

The hydrophilic amino acid and the hydrophobic amino acid referred toherein are variously defined depending on textbooks. In the presentinvention, as the hydrophilic amino acids, aspartic acid, glutamic acid,lysine, histidine, arginine and tyrosine were defined. Also as thehydrophobic amino acids, glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan and proline were defined.

In the present invention, as the hydrophilic amino acids, aspartic acid,glutamic acid, lysine, histidine, arginine and tyrosine were defined.Also as the hydrophobic amino acids, glycine, alanine, valine, leucine,isoleucine, methionine, phenylalanine, tryptophan and proline weredefined. However, it is thought that among the hydrophilic amino acids,His, Tyr, particularly Tyr are low hydrophilic, and among thehydrophobic amino acids, Gly, Ala, particularly Gly are low hydrophobic.Thus in order to predict more strictly, a good result is sometimesobtained when calculated by excluding Tyr and Gly, and more preferablyHis, Tyr, Gly and Ala. In modification of the blocking ability shownlater, it is preferable to modify with strong and weak of hydrophilicityand hydrophobicity in mind.

Thus subsequently, the content rates of respective amino acids werecalculated in various fragments. Consequently, it was demonstrated thatthe content rates of the hydrophilic amino acids and hydrophobic aminoacids tended to be different in each part obtained by dividing an aminoacid sequence of BSA (without signal peptide) or bovine a casein(without signal peptide) which exhibit the blocking ability into two,e.g., an N terminal side and a C terminal side (FIG. 14). In the presentinvention, a value obtained by dividing the content rate of thehydrophilic amino acids by the content rate of the hydrophobic aminoacids is conveniently defined as a “hydrophilic/hydrophobic rate”. Thehydrophilic/hydrophobic rate in the N terminal side of BSA is 1.00whereas it is 0.83 in the C terminal side. It is found that there is adifference of 0.17. In α casein, the value was 0.88 in the N terminalside whereas it was 0.64 in the C terminal side, and its difference was0.24. Meanwhile, in lipase derived from Pseudomonas, which does notexhibit the blocking ability, the value was 0.39 in the N terminal sidewhereas it was 0.44 in the C terminal side, and it was found that itsdifference was 0.05 which was less than 0.1. It was speculated from thisthat to exhibit the blocking ability, a hydrophilic region is requiredin addition to a relatively hydrophobic region in a protein molecule.

In the protein of the present invention, it is preferable that thehydrophilic/hydrophobic rate in a hydrophilic portion, a hydrophobicportion and full length are in the following ranges.

The hydrophilic/hydrophobic rate in the hydrophilic portion is 0.5 to2.0, more preferably 0.7 to 1.7 and still more preferably 0.8 to 1.5.

The hydrophilic/hydrophobic rate in the hydrophobic portion is 0.2 to1.1, more preferably 0.3 to 1.0 and still more preferably 0.4 to 0.9.

The hydrophilic/hydrophobic rate in the entire protein is 0.4 to 2.0,more preferably 0.5 to 1.5 and still more preferably 0.6 to 1.0.

The difference (absolute value) of the hydrophilic/hydrophobic rates inthe hydrophilic portion and the hydrophobic portion is preferably 0.1 to0.6, more preferably 0.15 to 0.5 and still more preferably 0.15 to 0.4.

The hydrophilic portion and the hydrophobic portion may be eitherdivided two, e.g., the C terminal side (or the N terminal side).

In the present invention, it is supposed that the hydrophilic portionand the hydrophobic portion are present in each portion of divided two,e.g., the C terminal region or the N terminal region. However, those inwhich the sequence which is neither hydrophilic nor hydrophobic has beenadded at N terminus or the C terminus are included in the presentinvention as long as the hydrophilic portion and the hydrophobic portionare present.

Furthermore in the present invention, as shown in FIG. 2, those havingrespective portions such as the hydrophilic portion-the hydrophobicportion-the hydrophilic portion, the hydrophobic portion-the hydrophilicportion-hydrophobic portion, and the hydrophilic portion-the hydrophobicportion-the hydrophilic portion-the hydrophobic portion in order arealso included in the present invention.

Based on the above, it is speculated that the hydrophobic portion(hydrophobic domain) is absorbed to the material surface and thehydrophilic portion (hydrophilic domain) is hovered over it, and thatviewed as whole, the blocking is performed by covering the materialsurface with the hydrophilic portion. That model is shown in FIG. 3. Itis predicted that lipase did not exhibit the blocking ability becauselipase was absorbed but the absorbed protein is too hydrophobic andfurther non-specific absorption to the protein occurred.

For the rate of the hydrophobic portion absorbed to the material surfaceand the hydrophilic portion which covers the hydrophobic portion, thehydrophilic portion is 0.3 to 10, preferably 0.5 to 5 and morepreferably 0.7 to 2 based on 1 of the hydrophobic portion. When thehydrophobic portion is too large, the hydrophilic portion can not coverit, and another protein is further absorbed to the hydrophobic portionabsorbed to the material surface. When the hydrophilic portion is toolarge, an area of the hydrophobic portion absorbed to the materialsurface becomes too small, the hydrophobic portion can not besufficiently firmly absorbed and the blocking efficiency is reduced.

In the present invention, “dividing into two” does not mean that thetotal sequence is divided into about 50% and 50%, and means that thesequence is divided into two of the hydrophobic portion and thehydrophilic portion. When one of or both of the hydrophilic portion andthe hydrophobic portion are plurally present, thehydrophilic/hydrophobic rate of the multiple hydrophilicportions/hydrophobic portions is calculated as an average value. Thehydrophilic portion and the hydrophobic portion have a clusteredportion, preferably a domain structure, respectively. Each portion is20% or more, preferably 30% or more and more preferably 40% or more ofthe total sequence. When the structure of the protein is unknown or isdifficult to be predicted, it is preferable and effective to analyze bybroadly dividing the amino acid sequence into the N terminal side andthe C terminal side.

As far as looking at FIG. 14, slight variation in difference of thehydrophilic/hydrophobic rates between the hydrophilic domain and thehydrophobic domain is observed among the proteins, and it seems thatonly the size of the value is not potentially reflect the blockingability in detail. One of the factors appears to be a molecular weight.In the present invention, “composed of more than 100 amino acidresidues” means that the total number of the amino acid residues in thetotal amino acid sequence is more than 100. Preferably, the amino acidsequence is composed of more than 150 amino acid residues, and morepreferably more than 200 amino acid residues. An upper limit ispreferably 2,000 amino acid residues, more preferably 1,500 amino acidresidues and still more preferably 1,000 amino acid residue. Thehydrophilic portion or domain is composed of preferably 30 or more aminoacid residues, more preferably 50 or more amino acid residues, stillmore preferably 60 or more amino acid residues, particularly preferably80 or more amino acid residues, and preferably 1,000 or less amino acidresidues and more preferably 500 or less amino acid residues.

The hydrophobic portion or domain is composed of preferably 30 or moreamino acid residues, more preferably 50 or more amino acid residues,still more preferably 60 or more amino acid residues, particularlypreferably 80 or more amino acid residues, and preferably 1,000 or lessamino acid residues and more preferably 500 or less amino acid residues.

As used herein, the domain refers to a region having structural orfunctional one cohesiveness in the molecule, and in the presentinvention the domain mainly refers to a unit having the structuralcohesiveness.

Subsequently, we tried to prove whether this hypothesis is correct ornot using a simple protein. A substrate-binding domain of HSP70 (DnaK),one type of heat shock proteins in Escherichia coli was used for thisexperiment. The structure of this protein (DnaK 384-607) has beenalready demonstrated by NMR analysis (FIG. 4), and it has been foundthat the protein is composed of two structural regions (domains), i.e.,a β sheet region (domain) in the N terminal side and an α helix region(domain) in the C terminal side. It has been also shown by calculationthat the hydrophilic/hydrophobic rate is 0.5 in the N terminal side and0.89 in the C terminal side, and that their difference is 0.39 which isa high value.

First, it was confirmed that this protein (DnaK 384-607) exhibited theblocking ability, consequently the protein was confirmed to have theblocking ability not as good as BSA (FIG. 5), and the availability ofthe present invention was shown.

Subsequently, various deletion mutants of DnaK 384-607 were made, and itwas examined what structure of DnaK played an important role forblocking. The mutants examined this time were shown in FIG. 6. Theexamined proteins are 6 types of DnaK 384-638, DnaK 384-607, DnaK384-578, DnaK 384-561, DnaK 508-607 and DnaK 525-607. These proteins canbe produced on a large scale using Escherichia coli as a host, and it ispossible to express by adding a histidine tag to the N terminal side ofthe protein, easily purify using a nickel chelate column and use for theexperiment. In the experiments this time, the protein to which thehistidine tag had been added was examined, but since the tag was small,its influence was ignored. Of course, in the case without the tagsequence, it was confirmed that the same result was obtained (Example7).

As a result of the experiment, it was found that the blocking effect wassignificantly reduced in DnaK 384-578 and DnaK 384-561 obtained bydeleting a part of the α helix structure, DnaK 508-607 and DnaK 525-607obtained by deleting the β sheet portion and seemed to be composed ofthe α helix structure alone (FIG. 5). Concerning DnaK 384-561, theconformational structure of DnaK 381-553 close thereto has been alreadyshown (FIG. 7), knowing it by analogy, it is predicted that the α helixstructure has been broken. The followings are suggested from these. Thatis, (1) the blocking ability is impaired by reducing or breaking thestructure of the hydrophilic portion in the protein having the blockingability. (2) The hydrophilic portion alone does not exhibit the blockingability. This seems to be because to exert the blocking ability, thehydrophilic portion is important in addition to the hydrophobic portionand it can be thought that the protein which exhibits the blockingability exerts the blocking effect by absorbing to the plate by thehydrophobic portion and covering the plate surface with the hydrophilicportion as shown in FIG. 3. It is predicted that this DnaK justcorresponds to a left figure in FIG. 3.

It is generally known that the hydrophilicity is lost by collapsing asecondary structure by denaturation or mutation. It is predicted that itis not suitable to prepare a partial sequence protein by breaking thehydrophilic domain as also shown from the above examples of DnaK. On thecontrary, it seems to be possible to enhance the hydrophobicity bybreaking the hydrophobic domain as shown in FIG. 8.

In the present invention, it seems that the domain is important and thatat least more than one domain structures are required. Thus, 100 aminoacid residues required for composing one structural domain is a roughstandard. Therefore, it can be said that it is desirable that thepresent invention has the domain structure composed of at least morethan 100 amino acid residues, i.e., more than one domain structures.

Based on such a fact, the present invention provides a method forscreening a novel protein or a novel partial sequence protein forblocking having the blocking ability based on amino acid sequence data.Particularly, the method is the method for screening the protein or thepartial sequence protein which meets the conditions shown below.

-   A) The amino acid sequence is divided into two, and an absolute    value of a difference between hydrophilic/hydrophobic rates in    divided two portions is 0.1 or more, calculated using the following    formula from content rates of hydrophilic amino acids (D, E, K, H,    R, Y) and hydrophobic amino acids (G, A, V, L, I, M, F, W, P);    [Hydrophilic/hydrophobic rate]=[Content rate of hydrophilic amino    acids]/[Content rate of hydrophobic amino acids].-   B) The hydrophilic portion (in higher value of    hydrophilic/hydrophobic rate) is 0.5 or more.-   C) The protein is composed of more than 100 amino acid residues.

In the above conditions, D is aspartic acid, E is glutamic acid, K islysine, H is histidine, R is arginine, Y is tyrosine, G is glycine, A isalanine, V is valine, L is leucine, I is isoleucine, M is methionine, Fis phenylalanine, W is tryptophan and P is proline. The hydrophilicamino acid and the hydrophobic amino acid are variously defineddepending on textbooks. In the present invention, the hydrophilic aminoacids were aspartic acid, glutamic acid, lysine, histidine, arginine andtyrosine. Also the hydrophobic amino acids were glycine, alanine,valine, leucine, isoleucine, methionine, phenylalanine, tryptophan andproline.

The partial sequence protein referred to herein indicates the proteincomposed of the partial amino acid sequence of the wild type protein andis preferably the protein having more than one domain structures.

When the amino acid sequence is divided into two of the N terminal sideand the C terminal side, it is not necessarily required to preciselydivide into two, but it is preferable to divide so that the numbers ofthe amino acid residues in both are almost equal as possible. When thesignal peptide is predicted from the amino acid sequence, it is betterto calculate by excluding that portion. In fact, in the presentinvention, in all of BSA, α-casein and lipase, the calculation wasperformed using the mature type of the amino acid sequence where thesignal peptide had been removed.

Division methods as shown in FIG. 2 other than the method of dividinginto two of the N terminal side and the C terminal side are preferablyused. In such a case, such a method can be preferably applied to theprotein whose conformational structure has been demonstrated orpredicted.

To calculate the amino acid contents from the amino acid primarysequence, it is effective to use software for analyzing gene/amino acidsequences. The analysis software is not particularly limited, and in thepresent invention, the analysis was performed using GENETYX (softwaredevelopment company).

It is also effective for efficiently performing screening works to makethe hydrophilicity and the hydrophobicity of the protein graphs using ahydrophobicity calculation function of the software. Preferably, acalculation formula of hopp & woods is used.

The present invention is the protein or the partial sequence proteinhaving the blocking ability, screened from the amino acid sequence bythe above method, and the protein or the partial sequence protein whichis obtained by the following analysis step D and meets the condition E.

-   D) 1. A step of adding a candidate protein (50 to 100 mg/mL, diluted    with 20 mM Tris-HCl, pH 7.0) which meets the conditions of claim 1    and bovine serum albumin (fraction V) prepared by the same way to    wells of a polystyrene immunotiter plate, blocking at 2 to 10° C.    for 4 to 5 hours and removing a liquid.-   2. A step of adding normal human serum diluted 25 to 100 times with    PBS(−), leaving stand at 37° C. for one hours, and subsequently    washing the plate with PBS(−) (0.05% Tween® (polysorbate)20).-   3. A step of comparing IgG amounts non-specifically absorbed to the    plate using an enzyme-labeled anti-human IgG antibody by a    calorimetric method using a chromogenic substrate.-   E) A developed color intensity for the candidate protein is 2.5    times or less, preferably 2 times or less, more preferably 1.5 times    or less, still more preferably 1.2 times or less and especially 1    time or less of the developed color intensity for bovine serum    albumin.

Herein, the blocking ability is measured by making non-specificabsorption of human serum IgG known for non-specific absorption to theplastic plate a rough standard. As used herein, the polystyreneimmunotiter plate (96-well type) refers to an immunotiter plate of96-well type made from polystyrene, generally used in immunoassays. Acandidate protein purified by an appropriate method is used, and finallydiluted to 50 to 100 mg/mL with 20 mM Tris-HCl (pH 7.0). Simultaneously,bovine serum albumin (BSA) is similarly prepared. As BSA, Fraction Vcommercially available from Sigma can be used. BSA need not always beFraction V as long as it has the same performance. The blocking isperformed at 2 to 8° C. for 4 to 5 hours, and subsequently the solutionis completely removed to progress to next step without washing.

The human serum used herein is not particularly limited as long as it isthe serum isolated from a normal adult. Since the undiluted serum is athigh concentration, it is better to dilute 25 to 50 times with PBS touse. Since the amount of serum IgG is different in individuals, adilution rate may be slightly changed. The non-specific absorption tothe plate is performed at 37° C. for 30 minutes. In order to preciselymeasure, it is preferable that the amount of the serum added at thistime is smaller than that of the solution used for the first blocking.For example, when 100 μL is used for the blocking, 50 μL of a dilutedserum solution is added. Finally, after reacting precisely for one hour,the solution is removed, and the plate is washed with the sufficientamount of 0.05% TWEEN® (polysorbate) 20/PBS(−). It is preferable to washthree times, and more preferably four times. Washing may be performedusing a plate washer for exclusive use.

Subsequently, an anti-human IgG antibody labeled with an enzyme isdiluted with an appropriate solution to an appropriate concentration,and added to the above wells. It is suitable to react at 37° C. for onehour. It is preferable to dilute the antibody to a level at which anordinary immunological detection is performed, and to use the solutioncontaining a suitable blocking agent (BSA or casein) for the dilution ofthe antibody because no variation is shown. More preferably, it isbetter to use 0.01 M phosphate buffer (pH 7.4) containing 0.15 M NaCland 0.5% casein. As the enzyme used for labeling, peroxidase and alkaliphosphatase are preferably used, and in particular, peroxidase ispreferably used. As a color development reagent for peroxidase,3,3′5,5′-tetramethylbenzidine (TMBZ) is preferably used. As a colordevelopment substrate for alkali phosphatase, WT-1 can be used. It isdesirable that a time period for color development is in the range atwhich an absorbance in the target well is quantitative (0.5 to 1.5).Note because no correct value is obtained when the absorbance exceedsthe quantitative range. No correct value is obtained when the plate isdried throughout all manipulation.

The lower the value obtained in this way is, it indicates the higherblocking efficiency. In the present invention, it was determined that asample had the blocking ability when the sample exhibited the valuewhich was 2.5 times or less than the value of BSA. Therefore, the novelprotein or the novel partial sequence protein for blocking of thepresent invention is the protein which meets the conditions shown inclaim 1 and exhibits the value 2.5 times or less than the value of BSAin this assay, i.e., having the blocking ability.

As an alternative method of this assay, it is also possible to use themethod shown in Example 7. That is, serially diluted peroxidase is addedto the candidate protein solution, which is then subjected to a solidphase to absorb for a certain time period, and subsequently thenon-specific absorption of peroxidase is measured. The absorbedperoxidase is confirmed as with the above method using the colordevelopment method of 3,3′,5,5′-tetramethylbenzidine. Also in thisassay, it is an indicator showing the blocking ability to exhibit thevalue 2.5 times or less than the value of BSA. Likewise, the dilutedperoxidase solution may be added to the solid phase previously blocked,and its non-specific absorption may be measured. In that case, it isalso the indicator showing the blocking ability to exhibit the value 2.5times or less than the value of BSA. The concentration of the peroxidasesolution used herein is preferably 0.05 mg/mL, but it is conductive tothe correct evaluation to control and use so as to measure in thecommonsense range. The dilution of peroxidase is preferably performedusing PBS(−), but is not particularly limited.

From the above, it can be said that it is preferable that the presentinvention has more than one domain structures, but as this example,especially for the hydrophobic domain, a part of the domain structure ofone in two domains may be lacked. It is preferable that the number ofthe amino acid residues in the protein or the partial sequence proteinof the present invention is 100 or more, preferably 150 or more and morepreferably 180 or more.

Furthermore, the present invention is the novel protein achieving theimproved blocking efficiency of the protein or the partial sequenceprotein by modifying the amino acid sequence of the protein which meetsthe conditions of the above claim 1. The “novel” referred to herein isthe “novel” at the level of amino acid modification, and the novelprotein may be the already known protein. In the case of the partialsequence, those having the partial sequence unknown so far are regardedas the novel proteins.

In the present invention, the amino acid sequence may be modified by anyof or a combination of substitution, deletion, and insertion. Fordirectionality of mutation, the mutation to further enhance thehydrophobicity in the hydrophobic region or the mutation to furtherenhance the hydrophilicity in the hydrophilic region are effective, andsuitably used. The deletion and the insertion of the amino acid sequenceare effective in order to change the conformational structure of theprotein, it is thought that the hydrophobic region hidden by thehydrophilic region is exposed on the protein surface. Meanwhile, asshown in the above example, it can be said that the amino acidmodification to break the hydrophilic domain structure is notpreferable.

The modified protein may be derived from the prokaryotic organism or theeukaryotic organism. Considering the mass production using Escherichiacoli, the protein derived from the prokaryotic organism is preferablyused. More preferably, the protein derived from Escherichia coli isused. The protein used may be a partial structure such as domain, whichis rather preferably used.

In particular, the blocking agent is often used as a stabilizing agentand an excipient by combining with an enzyme in some cases, and it canbe said that the protein which does not have a particular function of anenzymatic activity is suitable for the present invention. The proteinfrom which the domain having the enzymatic activity has been deleted ispreferably used.

In the present invention, the protein derived from HSP70 family proteinis preferably used. More preferably the protein derived from the DnaKprotein of Escherichia coli is used. Preferably, a substrate-bindingdomain of the HSP 70 family protein from which an ATPase domain has beendeleted is preferably used, and more preferably, the substrate-bindingdomain of the DnaK protein of Escherichia coli from which an ATPasedomain has been deleted is used. The substrate-binding domain of theDnaK protein was already introduced above as Example.

The protein belonging to the HSP70 family used for the present inventionis not particularly limited, and is selected from DnaK in Escherichiacoli, Ssalp present in yeast cytoplasm, Ssclp present in yeastmitochondria, Kar2p present in yeast endoplasmic reticulum, HSP70present in mammalian cytoplasm, Bip present in mammalian endoplasmicreticulum, mHsp70 present in mammalian mitochondria and HSC70 which isconstitutively expressed regardless of the presence or absence of heatshock and is a homolog of HSP70, and the like. Many homologs are knownin the HSP 70 family, and the above ones are parts of them. Of course,it is possible to easily predict that the same effect can be expectedfor homologs other than those listed above.

In particular DnaK in Escherichia coli has been well-studied, and thus,it can be said that it is relatively easy to predict the effect of theamino acid modification. This protein is composed of 638 amino acidresidues, and comprised of an “ATPase (ATP-binding) domain” composed ofthe amino acids at positions 1 to 385 and a “substrate-binding domain”composed of the amino acids at positions 386 to 638 (FIG. 6). Thesequence of the DnaK protein is shown in SEQ ID NO:1, and its genesequence is shown in SEQ ID NO:2. HSP70 has been known to bind to anewly formed polypeptide and a partially folded protein and helprefolding of the protein which can not fold spontaneously. It is alsoknown that HSP 70 recognizes abundant substrates because it works in arelatively early phase of protein synthesis.

It is also known that the substrate-binding domain itself of DnaK has anaction to persuade the refolding of the denatured protein (Proc. Natl.Acad. Sci. USA, 99:15398-15403, 2002), and it is thought that it ispossible to develop various intended uses by the use of this molecule.

When achieving the present invention, we focused this substrate-bindingdomain of DnaK, and could obtain various findings for expressionmechanism of blocking action by various examinations to complete thepresent invention.

First, we attempted to make the protein excellent in blocking efficiencyby adding the mutation to a β sheet structure portion to enhance thehydrophobicity in the β sheet portion. The attempt is that (1) a morehydrophobic portion of the β sheet is exposed by deleting a part of theN terminus of the β sheet (the β sheet structure is broken to enhancethe hydrophobicity) and (2) a hydrophilic amino acid in the β sheet issubstituted with a hydrophobic amino acid. As a result, remarkableenhancement of the blocking efficiency could be observed in DnaK 419-607where the N terminal portion of the β sheet structure had been deletedand DnaK 384-607 (D479V, D481V) where the hydrophilic amino acid hadbeen substituted with the hydrophobic amino acid (FIG. 9). This timeespecially, the remarkable enhancement of the blocking efficiency wasobserved in DnaK 419-607. It appears that this protein exhibits theblocking in a form shown in FIG. 8. That is, it is thought that bychanging the structure of the hydrophobic domain, the structure of thehydrophobic domain was changed to enhance the hydrophobicity of thehydrophobic domain, which was conductive to the enhancement of theblocking ability.

The enhancement of the blocking ability referred to in the presentinvention indicates that the blocking ability is further enhancedcompared to that of the original protein and protein partial sequence bygiving amino acid substitution, deletion and insertion to the candidateprotein.

This enhancement of the blocking ability is preferably measured by themethod shown in claim 2. The enhancement of the blocking abilityincludes the enhancement of the blocking ability for a long time periodand a short time period, and the blocking ability may be enhanced ineither one or both.

Discussing the reason why the efficiency was remarkable in DnaK 419-607in more detail, it is speculated that the efficiency is caused byexposing outside a part of an inside of a horseshoe shaped structurewhich is hydrophobicity-rich for trapping a peptide as a substrate of achaperone by removing the β sheet portion adjacent to a hinge portion ofthe horseshoe shaped structure of the DnaK substrate-binding domain.

That is, the present invention is the DnaK protein whose blockingefficiency has been improved, characterized by deleting the amino acidsequence from the N terminus to at least position 387 and at mostposition 472. The present invention is also the DnaK protein whoseblocking efficiency has been improved, characterized by deleting theamino acid sequence from the N terminus to at least position 387 and atmost position 418. More preferably, the present invention is the proteincomposed of the amino acid sequence at positions 419 to 607 in the DnaKprotein.

The present invention is the protein whose blocking efficiency has beenimproved, characterized in that the hydrophilic amino acids of a part ofthe DnaK protein where the ATPase domain or a part thereof has beendeleted are substituted with the hydrophobic amino acids. Moreparticularly, the present invention is the protein where the amino acidsequence in the part of the DnaK protein has been deleted by deletingthe ATPase domain or the part thereof, the protein where the blockingefficiency has been improved by substituting aspartic acid at positions479 and 481 with valine. More preferably, the protein composed of theamino acid sequence at positions 384 to 607 in the DnaK protein whereaspartic acid at positions 479 and 481 have been substituted with valineis used.

The modification referred to in the present invention indicates that theamino acid sequence has been modified by amino acid substitution,deletion and insertion in the candidate protein. For this modification,it is preferable to modify the amino acid by intending so that (1) thehydrophilic domain is not broken and (2) the hydrophobic domain is mademore hydrophobic, based on the above discussion after selecting thepartial sequence of the candidate protein or the protein. Although it isnot shown in the present invention, it should be of course considered tomake the hydrophilic domain more hydrophilic.

In the present invention, a blocking speed was also examined, andusefulness of the present invention was shown. That is, the blockingefficiency was measured when blocked for 5, 10, 30 minutes and 3 hours.As a result, it was found that DnaK 419-607 whose effect was the mostremarkable had the blocking efficiency of 87.5% whereas DnaK 384-638 hadthe blocking efficiency of 58.8% in blocking for 5 minutes. This speedis fast even compared with BSA which shows the equivalent effect after 3hours, and it is predicted that DnaK 419-607 is absorbed to thepolystyrene plate to exert the blocking effect at a very early stage.Therefore, by the use of this invention, it seems that it is possible todevelop a novel blocking agent having a performance equivalent or morethan that of conventional BSA.

The present invention is the protein for blocking having one or morehydrophilic domains and one or more hydrophobic domains, and the proteinfor blocking where the hydrophobic domain can be absorbed to thematerial surface and the hydrophilic domain can cover the hydrophobicdomain absorbed to the material surface. The domain referred to hereinis preferably a structural amino acid cluster composed of 50 or moreamino acids, and those partially deleted or added are regarded as thedomain.

The present invention also defines the blocking speed. That is, thepresent invention is the modified protein characterized in that theblocking speed is enhanced compared with that of BSA. Particularly, thepresent invention is also the modified protein characterized in that theblocking ability for less than 10 minutes is more excellent than that ofBSA under the condition where the protein amounts have been adjusted sothat the blocking efficiency equivalent to that of BSA is exhibited inblocking for 3 hours. The modification herein indicates that a genesequence encoding the protein of a wild type has been converted by aminoacid substitution, deletion and insertion. An evaluation method is notparticularly limited, methods shown in Examples 5 and 7 are preferablyused, and the blocking is measured using non-specific absorption of IgGor peroxidase to the polystyrene plate as the indicator. In theevaluation method using IgG, the blocking is performed for 1 to 10minutes, preferably 2 to 10 minutes, subsequently human serum dilutedwith PBS(−) is added, then washing is performed, an anti-human IgGantibody at an optimal concentration is reacted, subsequently washing isperformed, and then the amount of non-specifically absorbed IgG ismeasured by absorbance of colored products from TMBZ. Also in theevaluation method using peroxidase, first, horseradish peroxidase forlabeling (supplied from Toyobo Co., Ltd., PEO-131) is dissolved at 2mg/mL in the protein solution whose blocking ability is to be measured.Then serial dilutions of 40 to 320 times of the peroxidase solutions aremade from the above solution, and 100 μL of each dilution is dispensedin a polystyrene 96-well microplate. The plate is left stand at roomtemperature for one hour, subsequently the solution is removed, and theplate is washed with PBS buffer containing 0.02% TWEEN® (polysorbate)20. This washing manipulation is repeated six times, and then thewashing solution is thoroughly removed. Tetramethylbenzidine solution isadded, the plate is incubated at 37° C. precisely for 10 minutes, andthen 1N sulfuric acid is added to stop the reaction and develop thecolor. This absorbance is measured by a microplate reader at a majorwavelength of 450 nm and minor wavelength of 650 nm. Details will bedescribed in Example 7. Likewise, the diluted peroxidase solution may beadded to the solid phase previously blocked, and its non-specificabsorption may be measured. The concentration of the peroxidase solutionused herein is preferably 0.05 mg/mL, but it conductive to the correctevaluation to control and use so as to measure in the commonsense range.The dilution of peroxidase is preferably performed using PBS(−), but isnot particularly limited.

In this Example, as BSA which exhibits the blocking ability equivalentto that of 0.7 mg/mL of DnaK 419-607 after 3 hours, 2.4 mL/mL of BSA(Fraction V, supplied from Sigma, code: A-4503) was used (the proteinamount was measurement by Bradford method). The amount of the specimenprotein may be in the commonsense range for measuring the blockingability, and is preferably used in the range of 0.5 to 1 mg/mL. Theconcentration of BSA used for this measurement is the amount at whichthe blocking efficiency equivalent to that of the specimen protein after3 hours is exhibited, and this requires the previous examination of theconcentration. As the buffer which dissolves those proteins, 20 mMTris-HCl (pH 7.0) and PBS(−) are preferably used, but the buffer is notparticularly limited.

The protein or a protein fragment used for the present invention mayhave a tag, and in fact, the examination was performed using those wherea histidine tag had been added to the amino terminus. As the tag, any ofa histidine tag, a GST (glutathione S-transferase) tag, a MBP (maltosebinding protein) tag, a Flag tag, a Myc tag, and a TAP (tandem affinitypurification) tag may be used, and may be used by fusing an optionalprotein as needed. An optional amino acid sequence may be added to theknown tag.

Further more, it is a matter of course, and the tag unknown generally orthe optional amino acid sequence may be added. Concretely, when thepartial sequence of the protein whose N terminal side has been deletedis used, it is of course necessary to introduce a methionine residue tothe N terminus. Also considering the expression enhancement or therelation with restriction enzyme sites, the addition of several aminoacids is also thought. For example, four amino acids of MRGS have beenadded to the N terminus of DnaK 419-607N in Example 7. Of course, theprotein may be expressed as a fusion protein with an optional protein.The position of addition may be the N terminal side or the C terminalside.

An expression method of the present protein is not particularly limited,but the method of expressing using a prokaryotic organism is preferableand furthermore the method of expressing using Escherichia coli is morepreferable. An expression vector is not particularly limited, and thosegenerally used for the expression may be used.

Concerning the method of purifying the protein of the present invention,the protein can be efficiently purified in some cases by heating a crudepurification solution at an appropriate temperature and purifying itscentrifugation supernatant, in addition to the method of purifying usingthe above tag. The heating is performed preferably at 50° C. or aboveand more preferably 70° C. or above. The substrate-binding domain ofDnaK used this time has heat resistance, no aggregation is observedunder the condition at 70° C. for 30 minutes, and it has been confirmedthat the protein remains in the centrifugation supernatant fraction.Therefore, when purified, the protein can be easily purified bypurifying this heated centrifugation supernatant on columnchromatography, and it is very economical. It can be also anticipatedthat co-existing enzymes are deactivated by passing through the heatingstep.

The protein of the present invention whose blocking efficiency has beenimproved can be practically applied to blocking agents, excipients,stabilizing agents, refolding aids, coating agents for cell attachmentand coating agents for medical use. In particular, the protein isgreatly expected as the blocking agent in detection systems which takeadvantage of immune reactions. It is predicted that it is possible touse for ELISA, immunohistological staining and Western blotting. Infact, when the present invention was completed, a potential applicationto ELISA (enzyme-linked immunosorbent assay) was examined and actualgood results were obtained (FIG. 12).

One embodiment of the present invention is the blocking agent, theexcipient, the stabilizing agent or the refolding aid containing DnaK419-607.

One embodiment of the present invention is the blocking agent, theexcipient, the stabilizing agent or the refolding aid containing DnaK384-607 (D479V, D481V).

The effects of the present invention will be made more clearly byexemplifying Examples of the present invention.

EXAMPLE 1 Screening of Candidate Proteins

Candidate proteins were screened from amino acid sequences using mainlya nucleic acid/amino acid sequence analysis software: GENETYX (softwaredevelopment company). This software has a function to calculate thenumbers of hydrophilic amino acid residues and hydrophobic amino acidresidues from a primary sequence of the amino acids, and was convenient.The numbers of the hydrophilic amino acids and the hydrophobic aminoacids contained in the sequences of an N terminal side half and a Cterminal side half of the proteins found to exhibit various blockingability, the proteins derived from Escherichia coli or the partialsequence thereof were calculated by GENETYX, and hydrophilic amino acidcontent rates, hydrophobic amino acid content rates,hydrophilic/hydrophobic rates and absolute values of differences of thehydrophilic/hydrophobic rates between divided two are collectively shownin FIGS. 13 and 14. In accordance with the definition of GENETYX herein,the hydrophilic amino acids were aspartic acid, glutamic acid, lysine,histidine, arginine and tyrosine. The hydrophobic amino acids wereglycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan and proline.

EXAMPLE 2 Cloning and Expression of DnaK Fragments

A DnaK fragment was cloned by amplifying an objective gene fragmentusing PCR method with genomic DNA extracted from Escherichia coli K-12strain as a template. KOD-Plus-supplied from Toyobo Co., Ltd. was usedfor PCR amplification of the gene. Concretely, in the amplification ofDnaK 384-638, a sample was prepared to contain reaction buffer, 1 mMMgSO₄, 15 pmole primers shown in SEQ ID NOS:3 and 4, 1 unit ofpolymerase and 100 ng of Escherichia coli DNA in 50 μL of a reactionsolution, and subjected to the reaction of 25 cycles at 94° C. for 15seconds, 55° C. for 30 seconds and 68° C. for one minute after thereaction at 94° C. for 2 minutes. An amplified DNA fragment was digestedwith a restriction enzyme BamHI, and cloned into a BamHI-SmaI site ofpQE30 (the DNA fragment amplified using KOD-Plus-(supplied from TOYOBO)had a blunt end, and thus a downstream side of the amplified fragmentwas directly used). The sequence of the cloned gene was confirmed bysequencing analysis. A 6×His sequence (histidine tag) can be added tothe N terminus of the objective protein by cloning into this vector. Anexpression plasmid, pQE-DnaK 384-638 was made in this way.

Expression and purification of the protein were performed using asupernatant obtained by culturing JM109 in which the gene had beenintroduced in LB medium with shaking for 16 hours, collecting microbialcells by centrifugation, suspending the microbial cells in 20 mMTris-HCl (pH 7.0), to which sonication was then given, and subsequentlycentrifuging by high speed microcentrifuge. Concretely, the protein waspurified using His-Select HC Nickel Affinity Gel (supplied from Sigma),and was finally dialyzed overnight against 20 mM Tris-HCl (pH 7.0) touse for experiments. The protein concentration was measured by Bradfordmethod (Bio-Rad Protein Assay Kit: 500-0006, supplied from Bio-Rad)using BSA as the standard.

EXAMPLE 3 Preparation of C Terminus-Deleted DnaK Clone and Point Mutant

C terminus-deleted DnaK clones were made by using pQE-DnaK 384-638 madein Example 2 as the template and introducing a stop codon at an optionalposition using QuickChange method. Actually, the clones were made usingQuickChange site directive mutagenesis kit (supplied from Stratagene) inaccordance with instructions thereof. The produced clones and thecombination of the primers used are as follows. DnaK 384-607: SEQ IDNOS:5 and 6, DnaK 384-578: SEQ ID NOS:7 and 8, and DnaK 384-561: SEQ IDNOS:9 and 10. For DnaK 384-607 (D479V, D481V), mutations were introducedusing pQE-DnaK 384-607 as the template and SEQ ID NOS:11 and 12. Thesequence of each mutant was confirmed by sequencing analysis, andsubsequently the protein was purified in accordance with the methoddescribed in Example 2.

EXAMPLE 4 Preparation of N Terminus-Deleted DnaK Clones

The PCR amplification was performed using pQE-DnaK 384-607 produced inExample 2 as the template and the primers shown below, and an amplifiedfragment was introduced into the BamHI-SmaI site of the pQE30 vector inaccordance with the method in Example 2. The produced clones and sets ofthe primers are as follows. DnaK 508-607: SEQ ID NOS:13 and 4, DnaK525-607: SEQ ID NOS:14 and 4, and DnaK 419-607: SEQ ID NOS:15 and 4. Thesequence of each mutant produced was confirmed by sequencing analysis,and subsequently the protein was purified in accordance with the methoddescribed in Example 2.

EXAMPLE 5 Blocking Effect

The blocking efficiency was measured using the following method.

The examination was performed by making non-specific absorption of humanserum IgG to a polystyrene plate an indicator.

As the method, 100 μL of BSA (Fragment V, supplied from Sigma) andvarious DnaK samples diluted with 20 mM Tris-HCl (pH 7.0) were added toa polystyrene 96-well immunoplate (E.I.A./R.I.A. 8 Well Strip, suppliedfrom Costar), left stand at 4° C. for 4 hours (basically the plate wasleft stand for 4 hours, but when blocking time periods were examined,optional time periods were set.). Subsequently, the solution was removedfrom the plate, then 50 μL of normal human serum diluted with PBS(−) 50times was added, and incubated at 37° C. for one hour. Subsequently,each well was washed four times with 200 μL of a washing solution(PBS(−), 0.05% TWEEN® (polysorbate) 20), 50 μL of a peroxidase-labeledanti-human IgG antibody (supplied from Jackson ImmunoResearch) dilutedwith an antibody dilution (0.01 M PB (pH 7.4), 0.15 M NaCl, 0.5% casein)to an optimal concentration was added to each well, and incubated at 37°C. for one hour. Subsequently, each well was washed four times with 200μL of the washing solution (PBS(−), 0.05% TWEEN® (polysorbate)20), 100μL of a color development reagent (3,3′,5,5′-tetramethylbenzidine, TMBZ)was added to allow color development at room temperature for 5 minutes,and then 50 μL of 1N sulfuric acid was added to stop the reaction. Thecolor development was measured using an ELISA reader at a majorwavelength of 450 nm and a minor wavelength of 650 nm. The Bradfordmethod was used for quantification of the protein.

First, the blocking efficiency for 4 hours was measured for BSA, DnaK384-638, DnaK 384-607, DnaK 384-578, DnaK 508-607 and DnaK 525-607prepared at 0.7 mg/mL. Consequently as shown in FIG. 5, those other thanBSA, DnaK 384-638 and DnaK 384-607 exhibited the low blocking effect. Inthe clines which exhibited the low blocking effect, DnaK 384-607 andDnaK 384-578 are the clones where the α helix was deleted whereas DnaK508-607 and DnaK 525-607 are the clones where the β sheet was deleted.Therefore, it has been speculated that the blocking using thesubstrate-binding domain of DnaK requires both the α helix structure andthe β sheet structure.

Subsequently, the blocking effect for 4 hours was compared in DnaK384-607 (D479V, D481V) and DnaK 419-607 (each 0.7 mg/mL) made for thepurpose of enhancing the hydrophobicity of the β sheet using BSA andDnaK 384-607 at the same concentration as controls. As a result, bothDnaK 384-607 (D479V, D481V) and DnaK 419-607 exhibited higher blockingefficiency than BSA at the same concentration, and in particular theeffect was remarkable in DnaK 419-607 (FIG. 9).

Furthermore, blocking concentrations and the blocking effect in theblocking for 4 hours were examined for DnaK 419-607 which had thehighest effect in the above experiment, DnaK 384-638 and BSA. As aresult, 0.15 mg of DnaK 419-607 had more remarkable effect than 12 mg ofBSA, and DnaK 419-607 was suggested to be capable of being used as theblocking agent at very low concentrations (FIG. 10).

Finally, the blocking time period and the blocking effect were examinedfor DnaK 419-607 which had the highest effect in the above experiment,DnaK 384-638 and BSA. The protein concentration was 0.7 mg/mL for DnaK419-607 and DnaK 384-638, and was 2.4 mg/mL for BSA, at which BSAexhibited the effect equivalent to 0.7 mg of DnaK 416-607 in the aboveexamination. As a result, it was found that the time period required forthe blocking by DnaK 419-607 was dramatically short and the sufficientlyeffective blocking effect was obtained, and that DnaK 419-607 exhibitedthe blocking effect more early than BSA at 2.4 mg/mL which exhibited theequivalent effect after 3 hours (FIG. 11).

EXAMPLE 6 Blocking Effect (ELISA)

Usefulness of the DnaK fragments as the blocking agent was examinedusing an ELISA system for human carcinoembryonic antigen (hCEA). First,an anti-hCEA MoAb was diluted to 10 μL/mL with 50 mM carbonate buffer(pH 9.6), 100 μL of aliquot was added to a polystyrene immunoplate(E.I.A./R.I.A. 8 Well Strip, supplied from Costar), and then left standat 37° C. for one hour. After leaving stand, each well was washed threetimes with 150 μL of the washing solution (PBS(−), 0.05% TWEEN®(polysorbate) 20), subsequently 200 μL of a blocking solution was added,and left stand at 4° C. for 4 hours. As the blocking solution, 2.4 mg ofBSA (20 mM Tris-HCl, pH 7.0) generally used and DnaK 419-607 dissolvedin the same buffer were used, and 20 mM Tris-HCl (pH 7.0) alone wasadded as a blank. After removing the blocking solution, 50 μL of an hCEAsolution (Immunoflora supplied from Toyobo Co., Ltd.) diluted to 0, 2.5ng/mL and 5 ng/mL was added, the plate was incubated at 37° C. for onehour, and then washed four times with 150 μL of the washing solution.Subsequently, a peroxidase-labeled anti-hCEA antibody (Immunoflorasupplied from Toyobo Co., Ltd.) diluted to an optimal concentration wasadded, reacted at 37° C. for one hour, and the plate was washed threetimes with 150 μL of the washing solution (PBS(−), 0.05% TWEEN®(polysorbate) 20). Then, 100 μL of a substrate solution(3,3′,5,5′-tetramethylbenzidine, TMBZ) was added to develop a color at37° C. for 20 minutes with shielding light. Finally, 100 μL of areaction stop solution (1N H₂SO₄) was added, and the developed yellowcolor was measured at 450 nm/650 nm.

As a result, a linearity was lost in the blank whereas the result withgood linearity was obtained in DnaK 419-607 similarly to BSA, and it wasshown that DnaK 419-607 of the present invention could be sufficientlyapplied to the immunological assay (FIG. 12).

EXAMPLE 7 Comparison of Blocking Effects of (Native) DnaK FragmentsWithout Histidine Tag

Since all of the DnaK fragments constructed in Examples 2 to 4 have thehistidine tag derived from the expression vector pQE30 at the Nterminus, isoelectric points of the DnaK fragments shift to neutral pH.Thus, the DnaK fragments where the histidine tag had been deleted wereconstructed so as to keep the blocking ability owing to originalelectrostatic interaction.

The DnaK clone where the histidine tag had been deleted was made usingpQE-DnaK 419-607 made in Example 4 as the template and using QuickChangemethod to delete amino acids after an initiation codon to a DnaK regioncontaining the histidine tag. Actually, using the QuickChange sitedirective mutagenesis kit (supplied from Stratagene), a BamHI site wasintroduced upstream of the histidine tag. The manipulation was performedin accordance with instructions thereof. Primer sequences used at thattime are shown in SEQ ID NOS:16 and 17. As was also shown in Example 2,an upstream side of the cloned gene has been provided with the BamHIsite. Therefore, the clone where the histidine tag was deleted can beobtained by digesting the resulting vector with BamHI and recombining.

This expression vector was designated as pQE-DnaK 419-607N. The nativeDnaK 419-607 fragment was acquired by culturing transformants obtainedby transforming Escherichia coli JM109 with this pQE-DnaK 419-607N.

That is, Escherichia coli JM109 (pQE-DnaK 419-607N) was inoculated toterrific broth (1.2% polypeptone, 2.4% yeast extract, 0.5% glycerol, 17mM monopotassium phosphate, 72 mM dipotassium phosphate) containing 100mg/mL of ampicillin, and cultured at 32° C. for 20 hours with shaking.Microbial cells corresponding to 1 L of this culture were collected bycentrifugation, suspended in 200 mL of 100 mM Tris-HCl buffer pH 9.0,and disrupted by French press. Polyethylene imine was added at a finalconcentration of 0.1% to this disruption solution, heated at 60° C. for2 hours, and the supernatant was collected by centrifugation. Then,ammonium sulfate with 50% saturation was added, a precipitate wascollected by centrifugation, and re-dissolved in 100 mM Tris-HCl buffer,pH 9.0. Furthermore, the solution was heated at 64° C. for 14 hours, andthe supernatant was collected by centrifugation. This crude enzymesolution was subjected to SUPERDEX® 200 (supplied from AmershamBioscience) gel filtration chromatography, then ammonium sulfate with50% saturation was added, the precipitate was collected bycentrifugation, and re-dissolved in 100 mM Tris-HCl buffer, pH 9.0. Thesolution was desalted by gel filtration chromatography using SEPHADEX®G-25 (supplied from Amersham Bioscience) equilibrated with the samebuffer. Subsequently, this DnaK fragment was subjected to the columnchromatography using PHENYL SEPHAROSE™ Fast Flow equilibrated with 100mM Tris-HCl buffer pH 9.0 containing 20% saturated ammonium sulfate, andeluted with a linear gradient of 20 to 0% of saturated ammonium sulfateto acquire a purified DnaK fragment fraction. This purified fragmentfraction was condensed by ultrafiltration, and desalted with distilledwater to acquire the finally purified DnaK fragment.

The blocking efficiencies of BSA and the native DnaK 419-607 fragmentwere measured and compared as follows.

First, horseradish peroxidase (PEO-131, supplied from Toyobo Co., Ltd.)for labeling was dissolved at 2 mg/mL in PBS buffer containing BSA orthe native DnaK 419-607 fragment. At that time, commercially availableBSA (fraction V) was prepared at the concentration of 2 or 10 mg/mL, andthe native DnaK 419-607 fragment was prepared at the concentration of0.1 or 0.5 mg/mL. Then, serial dilutions of 40 to 320 times of theperoxidase dissolution were made using the same solutions, and 100 μL ofeach dilution was dispensed to a polystyrene 96-well microplate. Theplate was left stand at room temperature for one hour, then the solutionwas removed, and the well was washed with 200 μL of PBS buffercontaining 0.02% TWEEN® (polysorbate)20. This washing manipulation wasrepeated six times, and subsequently the washing solution was thoroughlyremoved. Then, 100 μL of a tetramethylbenzidine solution (supplied fromBio-Rad) was added, incubated at 37° C. precisely for 10 minutes, and100 μL of 1N sulfuric acid was added to stop the reaction and developthe color. This developed color was measured by a microplate reader at amajor wavelength of 450 nm and a minor wavelength of 650 nm.

The results were shown in FIGS. 15 to 17. The native DnaK 416-607fragment at 1/20 concentration of BSA exhibited inhibition ofnon-specific color development equivalent to that of BSA, demonstratingthat the native DnaK 419-607 fragment exhibits the blocking abilityabout 20 times of BSA.

INDUSTRIAL APPLICABILITY

In accordance with the present invention, it becomes possible to easilyscreen the novel protein or the novel partial sequence protein havingthe blocking ability based on the amino acid sequence data, and producethe protein capable of mass production in Escherichia coli and achievingthe improved blocking efficiency. In the protein obtained in accordancewith the present invention, the blocking ability is high, and the simpleand reliable result is obtained compared with the conventional methods.The present invention greatly contributes to clinical diagnosis wherethe immunoassay is applied and medical practice fields.

1. A modified DnaK protein comprising SEQ ID NO: 1, except that: (a) theATPase domain of SEQ ID NO: 1 is deleted, and (b) a part of the β-sheetdomain of SEQ ID NO: 1 is deleted and/or at least one hydrophilic aminoacid in the β-sheet domain of SEQ ID NO: 1 is substituted with ahydrophobic amino acid in order to expose a hydrophobic inside of aβ-sheet domain of the DnaK protein, wherein the modified DnaK proteinhas improved blocking efficiency as compared to a DnaK proteinconsisting of an amino acid sequence from position 384 to a C terminusof SEQ ID NO:
 1. 2. The modified DnaK protein of claim 1, wherein theATPase domain is an amino acid sequence from an N terminus to position383 of SEQ ID NO:
 1. 3. The modified DnaK protein of claim 2, whereinthe part of the β-sheet domain is an amino acid sequence from position384 to at least position 418 and to at most position 472 of SEQ IDNO:
 1. 4. The modified DnaK protein of claim 2, wherein the part of theβ-sheet domain is an amino acid sequence from position 384 to position418 of SEQ ID NO:
 1. 5. The modified DnaK protein of claim 1, wherein anamino acid sequence from position 608 to a C terminus of SEQ ID NO: 1 isdeleted.
 6. The modified DnaK protein of claim 1, wherein the at leastone hydrophilic amino acid in the β-sheet domain is selected from thegroup consisting of aspartic acid, glutamic acid, lysine, and arginine.7. The modified DnaK protein of claim 1, wherein the hydrophobic aminoacid is selected from the group consisting of valine, leucine,isoleucine, methionine, phenylalanine, tryptophan, and proline.
 8. Themodified DnaK protein of claim 1, wherein the at least one hydrophilicamino acid is aspartic acid, and the hydrophobic amino acid is valine.9. The modified DnaK protein of claim 1, wherein the at least onehydrophilic amino acid is aspartic acid at positions 479 and 481 of SEQID NO: 1, and the hydrophobic acid is valine.
 10. A compositioncomprising the modified DnaK protein of claim
 1. 11. The composition ofclaim 10, wherein the composition is a blocking reagent, stabilizingagent, excipient, protein folding accelerator, protein refoldingaccelerator, coating agent for cell attachment, or coating agent formedical use.
 12. A method for producing a modified DnaK protein, themethod comprising: (a) deleting an ATPase domain of a DnaK proteincomprising SEQ ID NO: 1, and (b) exposing a hydrophobic inside of aβ-sheet domain of the DnaK protein comprising SEQ ID NO: 1 by deleting apart of the β-sheet domain of SEQ ID NO: 1 and/or substituting at leastone hydrophilic amino acid in the β-sheet domain of SEQ ID NO: 1 with ahydrophobic amino acid, so as to provide a modified DnaK protein,wherein the modified DnaK protein has improved blocking efficiency ascompared to a DnaK protein consisting of an amino acid sequence fromposition 384 to a C terminus of SEQ ID NO:
 1. 13. The method of claim12, wherein the ATPase domain is an amino acid sequence from an Nterminus to position 383 of SEQ ID NO:
 1. 14. The method of claim 13,wherein the part of the β-sheet domain is an amino acid sequence fromposition 384 to at least position 418 and to at most position 472 iofSEQ ID NO:
 1. 15. The method of claim 14, wherein the part of theβ-sheet domain is an amino acid sequence from position 384 to position418 of SEQ ID NO:
 1. 16. The method of claim 12, further comprising: (c)deleting an amino acid sequence from position 608 to a C terminus of SEQID NO:
 1. 17. The method of claim 12, wherein the at least onehydrophilic amino acid in the β-sheet domain is selected from the groupconsisting of aspartic acid, glutamic acid, lysine, and arginine. 18.The method of claim 12, wherein the hydrophobic amino acid is selectedfrom the group consisting of valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, and proline.
 19. The method of claim 12,wherein the at least one hydrophilic amino acid is aspartic acid, andthe hydrophobic amino acid is valine.
 20. The method of claim 12,wherein the at least one hydrophilic amino acid is aspartic acid atpositions 479 and 481 of SEQ ID NO: 1, and the hydrophobic acid isvaline.